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. 2016 Jun 16;30(9):3194–3201. doi: 10.1096/fj.201500089R

Preferential epigenetic programming of estrogen response after in utero xenoestrogen (bisphenol-A) exposure

Elisa M Jorgensen 1,1, Myles H Alderman III 1,1, Hugh S Taylor 1,2
PMCID: PMC5001514  PMID: 27312807

Abstract

Bisphenol-A (BPA) is an environmentally ubiquitous estrogen-like endocrine-disrupting compound. Exposure to BPA in utero has been linked to female reproductive disorders, including endometrial hyperplasia and breast cancer. Estrogens are an etiological factor in many of these conditions. We sought to determine whether in utero exposure to BPA altered the global CpG methylation pattern of the uterine genome, subsequent gene expression, and estrogen response. Pregnant mice were exposed to an environmentally relevant dose of BPA or DMSO control. Uterine DNA and RNA were examined by using methylated DNA immunoprecipitation methylation microarray, expression microarray, and quantitative PCR. In utero BPA exposure altered the global CpG methylation profile of the uterine genome and subsequent gene expression. The effect on gene expression was not apparent until sexual maturation, which suggested that estrogen response was the primary alteration. Indeed, prenatal BPA exposure preferentially altered adult estrogen-responsive gene expression. Changes in estrogen response were accompanied by altered methylation that preferentially affected estrogen receptor-α (ERα)–binding genes. The majority of genes that demonstrated both altered expression and ERα binding had decreased methylation. BPA selectively altered the normal developmental programming of estrogen-responsive genes via modification of the genes that bind ERα. Gene–environment interactions driven by early life xenoestrogen exposure likely contributes to increased risk of estrogen-related disease in adults.—Jorgensen, E. M., Alderman, M. H., III, Taylor, H. S. Preferential epigenetic programming of estrogen response after in utero xenoestrogen (bisphenol-A) exposure.

Keywords: endocrine disruptor, methylation, ERα binding, uterus

Bisphenol A (BPA) is an endocrine-disrupting compound (EDC) that is ubiquitous in modern environments as a result of its use in production of consumer and industrial plastics. Exposure to EDCs has been linked to disruption of normal development, in particular, alterations to hormonal signaling pathways that are necessary for adequate development of the reproductive tract. Effects of exposure differ throughout the lifecycle, with critical stages of differentiation being the most vulnerable to disruption by EDCs (1). In utero exposure to xenoestrogen EDCs alters the pattern of development in the female reproductive tract and can affect gene expression long after exposure has ended (2, 3). The best-characterized EDC is diethylstilbestrol, which is a nonsteroidal estrogen that was used from 1947 to 1975 to prevent pregnancy loss; however, it was later banned after it was noted that women who were exposed to it as fetuses had a higher incidence of vaginal clear cell adenocarcinoma and structural defects of the female reproductive system (4). BPA is another xenoestrogen found in consumer plastics, such as water bottles, baby bottles, and food preparation containers. BPA is also found in industrial items, such as medical devices, food can liners, dental sealants, thermal receipt paper, epoxy resins, and polycarbonate plastics (5). BPA exposure is prevalent such that 93% of Americans have detectable levels of BPA in their urine (5). The investigation into prenatal exposure to BPA is critical as it has been shown that pregnant women and their fetuses are nearly universally exposed (6).

In rodent models, in utero exposure to BPA can result in lasting changes to mammary glands and endometrium (7–9). Prenatal exposure to BPA is linked to advanced puberty, uterine hyperplasia, alteration to the uterine epithelial layer, altered mammary development, increased body weight, higher incidence of breast and prostate cancer, and altered reproductive function (7, 10–12). Given the importance of steroidal sex hormones, especially estrogen, in the etiology of these pathologies, we hypothesized that BPA could interfere with normal regulation and signal transduction by estrogen.

BPA is a weakly estrogenic compound and binds to estrogen receptors-α and -β (ERα and ERβ) with an approximate 104-fold lower affinity than estradiol (E2), and results in a similarly lower activity of ER in the regulation of estrogen-responsive genes (13, 14). As a result of this low ER affinity and activity, developmental effects of BPA cannot be explained by its estrogenic activity. One well-documented mechanism for these lasting effects is epigenetic programming (15, 16). Changes in DNA methylation can increase or decrease gene expression by interfering with binding of transcriptional activators and repressors. The best-studied mechanism for epigenetic programming is CpG island methylation (17–19). Here, we hypothesized that BPA exposure would lead to preferentially altered methylation of ERα binding genes, causing reprogramming specific to estrogen receptor target genes.

Previous work in our laboratory has demonstrated that in utero exposure to BPA leads to decreased methylation of Hoxa10 (2). Hypomethylation of the Hoxa10 promoter after in utero BPA exposure was shown to modulate the estrogen response of the Hoxa10 estrogen response element (ERE), the binding site for ERα (20). To determine whether ERα binding genes are preferentially affected globally, we performed both expression and methylation profiling to determine the specificity of the effect of BPA on ERα binding genes.

MATERIALS AND METHODS

Animal model

All animal experiments were conducted in accordance with the Yale University Animal Care Committee Guidelines. CD-1 male and female mice were obtained from Charles River Laboratories (Wilmington, MA, USA).

Female CD-1 mice (n = 12; 8 wk old) were mated with 8-wk-old male CD-1 mice. Detection of vaginal plug indicated day 1 of gestation. On day 9, pregnant dams were anesthetized via i.p. injection of ketamine/xylazine rodent anesthesia mix. A lower abdominal vertical incision was made, and an Alzet (Cupertino, CA, USA) model 1002 osmotic minipump loaded with either 5.0 mg/kg/d BPA (n = 6) or DMSO vehicle control (n = 6) was inserted into the peritoneal cavity as specified by manufacturer protocol. The dams were monitored at least daily postsurgically and during the first postnatal week.

At 2 wk of age (before weaning), half of the female offspring from each treatment group were killed, and the reproductive tracts of the pups were isolated. At 6 wk, all remaining female offspring were ovariectomized by using a lower abdominal vertical incision. At 8 wk, ovariectomized mice were treated with a single intraperitoneal injection of 300 ng E2 or DMSO vehicle and the uteri were dissected 6 h later.

A portion of the collected uterine tissue was fixed in formalin and embedded in paraffin for histologic and immunohistochemical analysis, whereas remaining tissue was used to isolate RNA and DNA from each offspring. Tissue was stored in 1 ml RNAlater (Qiagen, Valencia, CA, USA) at −80°C until RNA and DNA isolation.

Maternal and fetal serum BPA quantification

At 17 d of gestation, pregnant dams were euthanized, and blood was collected via the vena cava (N = 5). Fetal blood was collected from the carotid artery (N = 5). Serum (50 μl) was purified via addition of methanol and acetonitrile and centrifugation. Quantification occurred as previously reported by using HPLC–tandem MS, with each sample run in triplicate (12).

RNA isolation

Each sample was placed in 1 ml TRIzol solution (Thermo Fisher Scientific, Waltham, MA, USA), and total RNA was purified by using the Qiagen RNeasy Plus Mini kit according to manufacturer instructions.

Gene expression microarray

Samples obtained from mice at 8 wk were pooled by litter (n = 4). Total RNA was labeled and hybridized to a mouse BeadChip WG-6 expression microarray (Illumina, San Diego, CA, USA). Data was analyzed by using BeadStudio (Illumina). Analysis of mRNA expression microarrays was performed by using Partek software (Partek, St. Louis, MO, USA). All genes with a fold change of ≥2 and P < 0.05 were defined as significantly differently expressed. Additional pathway analysis was completed with use of Qiagen Ingenuity Pathway Analysis.

Quantitative real-time PCR and analysis

For each sample, 500 ng of total RNA was reverse transcribed in 20 μl reaction mixture by using an iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). Intron-spanning primers were designed by using Perl Primer. Quantitative real-time RT-PCR reactions were prepared by using iQ SYBR Green Supermix (Bio-Rad). Each PCR reaction consisted of the following: 1 μl cDNA template, 1 μl forward primer (1 μM), 1 μl reverse primer (1 μM), 9.5 μl nuclease-free H2O, and 12.5 μl iQ SYBR Green Supermix. PCR was performed for 40 cycles at 95°C for 15 s, at 58.7°C for 20 s, and at 72°C for 25 s. Bio-Rad iCycler iQ system was used to quantify fluorescence of PCR products during amplification. Melting curve data were collected for analysis. Specificity of amplified products and absence of primer-dimers were confirmed via melt curve analysis. All products obtained yielded the predicted melting temperature. Gene expression was normalized to the expression of β-actin for each sample. Relative mRNA expression of each gene was calculated by using the 2−ΔΔCT method (21). Data were analyzed by either Student’s t test or ANOVA with Tukey multiple comparisons post hoc testing. All experiments were conducted in triplicate using samples from 6 mice in each group, with each mouse being from a different litter. Samples without cDNA template were used as negative controls.

DNA isolation

Tissue samples were thawed on ice. Total DNA was purified by using the Qiagen DNeasy Tissue Kit according to manufacturer instructions. Genomic DNA samples were stored at −80°C until further use.

Methylated DNA immunoprecipitation

Genomic DNA samples were fragmented and then enriched for methylated DNA as required for NimbleGen methylation arrays. Total genomic DNA (6 μg) was digested overnight in 24 U of MseI that was supplemented with 100 ng/μl bovine serum albumin. Reaction was stopped by heating to 65°C for 20 min, and then samples were purified by using a QIAquick PCR Purification Kit (Qiagen). Quality of digestion was assessed by running samples on a 2% agarose gel. MseI–digested DNA was heat-denatured and incubated overnight at 4°C with monoclonal mouse anti–5-methyl cytidine (Abcam, Cambridge, MA, USA). DNA–antibody mixture was then incubated for 2 h at 4°C with prewashed protein A-agarose beads. Beads were washed 3 times, resuspended in digestion buffer that contained proteinase K, and incubated overnight at 55°C. DNA was extracted by using organic extractions of phenol followed by chloroform/isoamyl alcohol. Aqueous layers were transferred to fresh tubes and DNA was precipitated with ethanol. The resulting pellet was air dried and resuspended in 10 mM Tris-HCl at pH 8.5. Immunoprecipitated and input DNA were then amplified by using a GenomePlex Complete Whole Genome Amplification Kit (Sigma-Aldrich, St. Louis, MO, USA) according to manufacturer instructions. Samples were then purified by using the QIAquick PCR purification kit.

Methylation array

Samples from mice at 2 wk of age were pooled by litter (N = 4). Genomic DNA was enriched by using methylated DNA immunoprecipitation (MeDIP), then labeled and hybridized to a mouse 720K CpG Promoter Methylation Array (Roche/NimbleGen, Madison, WI, USA). Statistical analysis of the MeDIP methylation microarray data was performed by using sliding window algorithm to pool neighboring probes together and then tested for differences within each sliding window. Windows with false discovery rate control (P < 0.05) are identified as differentially methylated regions.

ERα binding site methylation analysis

ERα binding sites were determined by using a preexisting uterine ERα chromatin immunoprecipitation sequencing data set from wild-type mice treated with E2 that was reported by Hewitt et al. (National Center for Biotechnology Information Gene Expression Omnibus accession GSM894054; http://www.ncbi.nlm.nih.gov/geo) (22). Alignment of sequencing data was performed using STARS to the mm10 genome, and annotation was created by using the Homer annotation system to the mm10 genome (23). Peaks with <30 reads were discarded and peaks with >30 reads were defined as ERα binding sites regardless of distance from known transcription sites or gene location. Methylation status of all genes in the methylation array was determined by the mean log2 florescence change of MeDIP microarray for the group of genes in question, either all genes on the array or only genes with ERα binding sites.

RESULTS

Serum BPA levels approximate human environmental exposure

Pregnant dams had a mean BPA serum concentration of 8.2 ng/ml from day 9 to day 21 of gestation (total of 12 d), which resulted in a fetal serum level of 7.52 ng/ml at day 17 of gestation. In nonpregnant humans, typical plasma BPA levels range from 0 to 2 ng/ml (25, 26). These values are higher in pregnant women, with an increase to 0.3–18.9 ng/ml (25, 27). In the human fetus, BPA values range from 0.2 to 9.2 ng/ml in fetal serum, as sampled from the umbilical vein (25, 28).

Adult mice show global changes to uterine gene expression after BPA treatment in utero

Before puberty, and therefore before any endogenous estrogen exposure, DNA and RNA were obtained from the uteri of half the female offspring at the age of 2 wk. DNA methylation analysis was performed using a MeDIP microarray, and RNA expression analysis was performed using an expression microarray. At 6 wk of age—after sexual maturation and estrogen exposure—remaining female offspring were ovariectomized. At 8 wk, ovariectomized mice were treated with E2 or vehicle control, and the uterine global expression profile was analyzed.

To determine whether global gene expression was affected by in utero BPA exposure before sexual maturation and, therefore, before increased levels of sex hormones, we analyzed the expression profile of uterine RNA from 2-wk-old pups. Sexually immature female offspring at 2 wk of life had remarkably similar uterine gene expression profiles, independent of their in utero BPA exposure. Only 18 of a total 45,000 gene sets tested displayed >2-fold difference in expression. Ten genes were up-regulated and 8 were down-regulated. (Fig. 1) These results indicate that, before endogenous estrogen exposure associated with puberty, in utero BPA exposure had little influence on uterine gene expression.

Figure 1.

Figure 1.

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Global expression microarray from uterine tissue of mice exposed to BPA or vehicle control in utero. Before sexual maturation (2 wk), only 18 of 45,000 genes tested showed a >2-fold difference in expression between BPA- and vehicle-exposed mice. After sexual maturation (8 wk), intact mice exposed to BPA in utero had 365 genes with expression that differed by >2-fold. Likewise, 313 genes showed significant changes in expression in ovariectomized animals in response to E2 treatment (n = 4). CTL, control.

To determine whether global gene expression after in utero BPA exposure was altered after sexual maturation, we analyzed uterine RNA of adult mice by expression microarray. To control for endogenous estrogens present after sexual maturation, we compared expression profiles of mice treated in utero with BPA or vehicle and subsequently ovariectomized and later injected with E2 or vehicle. After endogenous estrogen exposure after the time of puberty, adult offspring exposed to BPA in utero showed 365 genes that were differentially expressed >2-fold compared with those without prenatal BPA exposure. Seventy-seven genes were overexpressed, whereas 288 were underexpressed relative to vehicle-controlled cohorts (Fig. 1). Expression levels of 365 genes were permanently altered only after puberty, which indicated a long-lasting effect of BPA on many genes induced by exposure to endogenous estrogens at the time of puberty. After a 6-h E2 treatment, 90 genes were up-regulated and 226 genes were down-regulated. A total of 316 genes showed >2-fold alteration in their response (Fig. 1). We used pathway analysis to examine possible downstream effects and identify canonical pathways affected by in utero BPA exposure. Affected pathways are shown in Supplemental Table 1.

The difference in the number of genes differentially expressed in intact animals and those that were ovariectomized and administered E2 injections confirms that it is the estrogen response, rather than other aspects of aging or puberty, that has been altered by in utero exposure to BPA. The difference in the number of genes differentially expressed between 2-wk-old mice and 8-wk-old mice demonstrates that effects of in utero BPA exposure are exacerbated after sexual maturation. In utero BPA exposure reprogrammed the estrogen response globally. Levels of ESR1 and ESR2 were not significantly different as determined by microarray and quantitative PCR, which indicated that these changes were not mediated through programming of ERα or ERβ expression.

Altered estrogen response after BPA exposure

The differences in expression were validated by using quantitative PCR. We analyzed 21 genes, and all results were similar to expression microarray results. We found several trends that genes in animals exposed in utero to BPA displayed. These include up-regulation and down-regulation as well as loss of repression, gain of induction, and gain of repression by E2.

Several genes were significantly up-regulated in adult offspring prenatally exposed to BPA without endogenous or exogenous E2 treatment and compared with non-BPA–exposed controls. Representative genes included TGF-β induced (Tgfbi), stearoyl-CoA desaturase 1 (Scd1), and RET proto-oncogene (Ret) (Fig. 2A). Scd1 expression was increased 1.84-fold in adult mice treated with BPA in utero (P = 0.034) (29). Tgfbi expression was increased 2.44-fold (P = 0.045), and Ret expression was increased 2.36-fold (P = 0.008).

Figure 2.

Figure 2.

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Uterine gene expression was altered by in utero exposure to BPA, as verified by quantitative PCR. Data were analyzed by using Student’s t test. Error bars indicate sem. A) Representative examples of genes up-regulated in mice exposed to BPA in utero, including Tgfbi, Scd1, and Ret (P = 0.045, P = 0.034, P = 0.008). B) Representative genes showing down-regulation after in utero exposure, including Fbln2, Muc1, and Lcn2. (P = 0.034, P = 0.005, P = 0.04; n = 6). CTL, control. *P < 0.05; **P < 0.01.

Genes that were significantly down-regulated in the BPA-exposed offspring include fibulin 2 (Fbln2), mucin 1 (Muc1), and lipocallin 2 (Lcn2) (Fig. 2B). Fbln2 expression was decreased by 2.30-fold in adult mice treated in utero with BPA (P = 0.034), and Muc1 expression was decreased 3.63-fold (P = 0.005). Lcn2 expression was decreased 14.34-fold in BPA-exposed mice (P = 0.04).

Estrogen response of several genes was also altered by in utero BPA exposure. One type of altered estrogen response observed was loss of estrogen-induced repression, as demonstrated by Il1β, Mmp3, Mmp10, and S100A9 (Fig. 3A). These genes all showed significant repression after estrogen exposure in the control group; however, after in utero BPA exposure, there was no significant change between the control and estrogen-exposed state.

Figure 3.

Figure 3.

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In utero exposure to BPA affected E2-responsive gene expression as confirmed by quantitative PCR. On the x axis, the first term indicates prenatal exposure and the second term indicates subsequent adult treatment. Data were analyzed by ANOVA with Tukey post hoc tests. A) Examples of genes that lost t estrogen response include Il1β, Mmp10, S100a9, and Mmp3 (P < 0.0001, P < 0.0001, P < 0.0001, P = 0.0285). B) Examples of genes that displayed an exaggerated induction by E2 after in utero exposure to BPA include Dkkl1, Fzd10, Gdf10, Wif1, and S100A8 (P = 0.0003, P = 0.0068, P = 0.005, P = 0.028; n = 6). Error bars indicate sem. CTL, control. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

In addition, several genes were identified that showed a gain of estrogen induction after exposure to BPA in utero (Fig. 3B). These genes include Dkkl1, Fzd10, Gdf10, Wif1, and S100a8. Of particular note is Dkkl1, which normally is subject to significant repression by estrogens; however, after in utero BPA exposure, treatment with E2 induces expression of the gene paradoxically.

The final trend identified was the gain of estrogen-mediated repression. Representative genes, including Hsd11b2, Mfap2, G6pdx, Krt7, Col6a2 and Stat5a, displayed a gain of estrogen-mediated repression after in utero BPA exposure (Fig. 4).

Figure 4.

Figure 4.

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In utero exposure to BPA leads to a gain of estrogen-mediated repression after as confirmed by quantitative PCR. On the x axis, the first term indicates prenatal exposure and the second term indicates subsequent adult treatment. Results were analyzed by ANOVA with Tukey post hoc tests. Examples of genes that displayed a gain of E2-mediated repression after in utero exposure to BPA include HSd11b2, Mfap2, G6pdx, Krt7, and Col6a2 (P = 0.001, P = 0.002, P = 0.032, P = 0.01, and P = 0.01, respectively). Error bars indicate sem. CTL, control. *P < 0.05; **P < 0.01; ****P < 0.0001.

Mice demonstrate altered promoter methylation after BPA treatment in utero

To test our hypothesis that CpG island methylation was responsible for changes in ER response programming, we used a MeDIP methylation array to quantify the effect of in utero BPA exposure on promoter methylation between exposed and unexposed offspring (Fig. 5). We searched promoter regions between 3 kb upstream to 1 kb downstream of the transcription start site. In addition, we defined a differentially methylated region as a minimum of 7 differentially quantified probes (750 bp), with a maximum of 500 bp separation. By using these parameters, we determined that animals exposed to BPA had 1719 differentially methylated promoter regions comprised of 744 hypermethylated and 975 hypomethylated unique genes (Supplemental Table 2).

Figure 5.

Figure 5.

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Heatmap of methylation changes in mice exposed to BPA in utero. Mice at 2 wk showed altered methylation patterns, as shown by the clustering of 1,719 genes on the basis of their difference in methylation status between in utero BPA exposure and control groups (n = 4). CTL, control.

Altered methylation after in utero BPA exposure preferentially affects ERα binding genes

BPA exposure affects methylation of genes that bind to ERα preferentially compared with all genes. To determine whether BPA altered CpG methylation of ERα binding genes to a greater extent than all other genes, we used a preexisting ERα chromatin immunoprecipitation sequencing data set and applied it to the analysis of the MeDIP microarray (22). By comparing the mean change in fluorescence between 2 list of genes—those that bound ERα and all genes—we were able to determine whether subsets of genes were hypo-/hypermethylated compared with mean. Of genes with significant alterations to methylation status after BPA exposure, 93% (1599/1719) also bound ERα. Mean absolute log2 fluorescence ratio change for animals exposed to BPA in utero compared with control animals for all genes was 0.5 (95% CI 0.46–0.54), and for ERα binding genes, the mean ratio change was 0.32 (95% CI 0.18–0.46; P = 0.014) (Fig. 6). These data demonstrate that ERα binding genes had lower levels of methylation than did all other genes, which indicates that BPA preferentially targeted methylation of ERα binding genes. Furthermore, of 316 genes that were differentially expressed in groups exposed in utero to BPA (Fig. 1), 85 were also examined for differences in promoter methylation. The number of tested genes was limited to genes for which probes were located on both the expression microarray and the MeDIP microarray. Of those 85 genes that were on both arrays, 55 (65%) demonstrated methylation changes >2-fold in the promoter region.

Figure 6.

Figure 6.

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After in utero BPA exposure, ERα binding genes, as determined by chromatin immunoprecipitation sequencing, demonstrate hypomethylation compared with the average of all other genes. Methylation was determined by the mean absolute log2 fluorescence ratio change between all genes (0.5) and those genes capable of binding to ERα in animals exposed to BPA (0.35) (n = 4). Error bars indicate sem. *P = 0.014.

DISCUSSION

BPA is an environmentally ubiquitous synthetic molecule as a result of its use in the manufacturing of polycarbonates (5). Previous literature has linked prenatal exposure to BPA with a multitude of adverse outcomes, including prostate and breast cancers as well as altered reproductive function (2, 8, 9, 16, 30–32). In utero exposure to BPA has also been shown to affect the methylation pattern of individual estrogen-responsive genes, such as Hoxa10 (2, 33). We previously demonstrated that hypomethylation of the Hoxa10 ERE alters expression of the aberrantly methylated gene in response to E2 (3). Here, we examined the effect of in utero exposure to BPA on the global epigenetic regulation of gene expression in an estrogen-responsive organ. We demonstrated that BPA has a significant impact on the global expression and methylation pattern of uterine genes in mice exposed in utero, and we also demonstrated preferential hypomethylation of ERα binding genes.

Although these epigenetic changes associated with in utero exposure are likely present from birth, changes in gene expression did not manifest until sexual maturation with accompanying exposure to E2. We detected aberrant methylation in exposed pups at 2 wk of age before widespread changes in gene expression. In addition, we demonstrate that genes that bind to ERα are hypomethylated compared with all other genes at 2 wk. Although we have previously shown that hypomethylation of ERE in the Hoxa10 gene significantly changes its expression in response to E2, we show that this is a widespread phenomenon that affects a large number of estrogen-responsive genes (20). As gene expression was not manifest until sexual maturation, our findings suggest ERα-regulated genes are differentially and preferentially targeted by BPA. These changes in expression of estrogen-responsive genes can lead to altered expression of downstream target genes and can result in detrimental effects to the organism. Whereas the methylation state of the promotor region may change during sexual maturation, the significant overlap between prematuration aberrantly methylated genes and genes with altered postmaturation expression demonstrates that the in utero programming of those genes has an effect on gene expression postmaturation

BPA exposure in utero has several functional, estrogen-related outcomes, including, but not limited to, advanced puberty, altered ovarian function, decreased vaginal weight, infertility, endometriosis, endometrial hyperplasia, and breast cancer (9, 33). Our study provides a possible mechanism to explain why organisms exposed in utero to BPA often have diseases specific to the development of secondary sexual characteristics, the reproductive tract, and breast. Selective alteration of ERα binding genes leads to susceptibility to estrogen-induced disease.

To determine if there was any canonical pathway alteration, we performed canonical pathway analyses of genes differentially expressed between the control E2 and BPA E2 groups. We found 52 significantly altered pathways (Supplemental Table 1). It is noteworthy that many genes in neoplastic pathways had aberrant expression in adult mice that were exposed to BPA in utero. These changes in gene expression may contribute to the increased propensity for cancer in animals prenatally exposed to BPA. Several genes that contribute to extracellular matrix remodeling, including Mmp10, Mmp3, Fbln, Krt7, and Muc1, can contribute to increased tumor invasion. Aberration to cell cycle regulators, such as the S100s, can cause unchecked growth, and changes to the regulation of cell signaling components Ret, Stat5a, Fzd10, Cxcl1, and Wif1 can lead to uncontrolled growth and differentiation (24, 34–37). The abnormal response of these genes to E2 stimulation as a result of BPA treatment in utero is a particularly interesting link to the development of estrogen-sensitive cancers.

Whereas BPA preferentially affected methylation of many genes with known ERα binding ability, it also changed the methylation of some genes that do not bind to ERα. These alterations to the methylome may affect other pathways and have detrimental effects to offspring not associated with estrogen response or ERα binding. Changes in methylation may also prevent or enhance binding of corepressors and coactivators that can indirectly regulate genes that do bind to ERα. Alternatively, regulated genes without an ERα binding site may be regulated by a remote ERα outside of the promoter region and not yet on the annotation list. Furthermore, as a result of the MeDIP array having a different coverage than the expression array, we were not able to examine all estrogen-responsive genes in the uterus, and it is possible that that more genes are directly methylated by BPA exposure than identified; however, the high overlap between genes with altered methylation states and genes that bind to ERα demonstrates that there is a preferential effect of in utero BPA exposure on methylation of ERα binding genes. Our results likely underestimate the true effect of BPA exposure on gene methylation; however, they do indicate a targeted effect on methylation and expression of genes that bind ERα.

In conclusion, in utero exposure to BPA leads to permanent epigenetic changes in exposed offspring. Changes in methylation alter gene expression; however, this does not happen until sexual maturity when E2 levels rise. BPA preferentially programs ERα binding genes globally, thereby altering tissue response to E2 later in life. These changes likely contribute to estrogen-responsive diseases, such as endometrial hypo-/hyperplasia, uterine cancer, breast cancer, ovarian cancer, and infertility. Estrogen-responsive diseases may have their origin in fetal exposure to estrogen-like endocrine disruptors that alter DNA methylation.

ACKNOWLEDGMENTS

The authors thank Aiping Lin (Department of Biostatistics, Yale University) and Xiaoqing Yu (Department of Public Health, Yale University) for assistance with data analysis. This work was supported by U.S. National Institutes of Health (NIH) Eunice Kennedy Shriver National Institute of Child Health and Human Development Grant R01-HD076422; and NIH National Institute of Environmental Health Sciences Grant R01-ES010610. Author contributions: E. M. Jorgensen, M. H. Alderman, and H. S. Taylor designed the research, analyzed data, and wrote the paper. E. M. Jorgensen and M. H. Alderman performed the research. The authors declare no conflicts of interest.

Glossary

BPA

bisphenol-A

E2

estradiol

EDC

endocrine-disrupting compound

ER

estrogen receptor

ERE

estrogen response element

Fbln2

fibulin 2

Lcn2

lipocallin 2

MeDIP

methylated DNA immunoprecipitation

Muc1

mucin 1

Ret

RET proto-oncogene

Scd1

stearoyl-CoA desaturase 1

Tgfbi

TGF-β induced

Footnotes

This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.

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